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Transcript
Supplementary information
Table of contents
1.
Materials ............................................................................................................................ 2
1.1 Chemicals ........................................................................................................................ 2
1.2 Bacterial strains, plasmids and enzymes .......................................................................... 2
1.3 Purification of lipase T6M and fraction preparation for immobilization ................................ 2
1.4 Preparation of pET9a lysate ............................................................................................. 3
2.
Immobilization methods ................................................................................................... 3
2.1 Commercial lipases immobilization with and without pET9a crude lysate ......................... 3
2.2 Sol-gel immobilization ....................................................................................................... 4
3.
Activity assays .................................................................................................................. 4
3.1 Soluble lipase activity assay ............................................................................................. 4
3.2 Esterification activity assay for immobilized lipase ............................................................ 4
3.2 GC/MS analysis of butyl-laurate ....................................................................................... 5
4.
Biodiesel production tests ............................................................................................... 5
4.1 Enzymatic transesterification of soybean oil and waste chicken oil by sol gel immobilized
lipase ...................................................................................................................................... 5
4.2 Gas chromatography analysis of FAME............................................................................ 5
4.3 Recycling experiments of sol-gel immobilized lipase (CE) ................................................ 5
4.4 Scanning electron microscopy (SEM) analysis of sol-gel immobilized lipase .................... 6
References ............................................................................................................................... 6
1
1. Materials
1.1 Chemicals
Tetraethoxysilane (TEOS), dimethyldiethoxysilane (DMDEOS), phenyltriethoxysilane (PTEOS) and
octyltriethoxysilane (OTEOS) were purchased from Sigma–Aldrich (Rehovot, Israel). Lauric acid was
purchased from Acros Organics (NJ, USA) and n-butanol was purchased from Frutarum (Haifa,
Israel). NaF was purchased from Merck (Darmstadt, Germany) and imidazole from Alfa-Aesar
(Lancashire, England). Methanol, glycerol, n-hexane, NaCl and Triton X-100 were purchased from
Bio-Labs (Jerusalem, Israel) and 2-propanol from J.T. Baker (Deventer, The Netherlands). Ethyl
acetate was purchased from Gadot (Haifa, Israel) while acetonitrile, polyethylene glycol (PEG 1000)
and CaCl2 from Spectrum Chemical MFG (Gardena, CA, USA). Trizma-base, 4-nitrophenyl laurate
(pNPL), butyl laurate, heptadecanoic acid methyl ester and kanamycin were purchased from
SigmaAldrich (Rehovot, Israel). p-Nitrophenol and skim milk (SM) powder were purchased from
Fluka (Buchs, Switzerland). Refined soybean oil was purchased from a local grocery store, and
waste chicken oil was obtained from Green City Ltd. (Ein Hamifratz, Israel). All materials used were
of the highest purity available.
1.2 Bacterial strains, plasmids and enzymes
Recombinant Geobacillus stearothermophilus T6M lipase (EMBL, AF429311.1) fused to a His-tag
was expressed in Escherichia coli BL21 cells (DE3; Novagen, Darmstadt, Germany) as previously
described [(17)]. In addition, E. coli BL21 cells harboring pET9a vector only (without the lipase T6M
gene) were used as a control (16, 17). Novozymes® Lipolase 100L (Thermomyces lanuginosus
lipase, TLL) and CaLB (Candida antarctica lipase B) were purchased from Sigma–Aldrich (Rehovot,
Israel) as soluble lipase solutions.
1.3 Purification of lipase T6M and fraction preparation for immobilization
Lipase T6 variant H86Y/A269T/R374W (lipase T6M) with a His-tag was purified in a one-step process
using a Ni(II)-bound affinity column (HisTrap HP, Amersham Biosciences, Giles, UK). After overnight
growth of E. coli BL21 (DE3) cells harboring lipase T6M in 0.5L TB medium (enriched with 0.5 mM
CaCl2) with 25 μg ml−1 kanamycin, the cells were harvested by centrifugation (8,000×g for 10 min at
15 °C) and resuspended in a binding buffer (20 mM Tris–HCl buffer pH 7.5, 0.5 mM CaCl2, 0.1 mM
Triton X-100, 500 mM NaCl, and 20 mM imidazole). The cells were broken using a homogenizer
(EmulsiFlex-C3 High Pressure Homogenizer, AVESTIN, Ottawa, Canada) followed by centrifugation
(16,000×g for 20 min at 15 °C) for the removal of cell debris. The supernatant after this step, which
was defined as cell extract (LipT6M_CE fraction) was collected for immobilization and in parallel
separated and exposed to heat treatment of 50 °C for 15 min followed by another centrifugation step.
The supernatant after this step, defined as heat-treated CE (LipT6M_HT fraction) was collected for
immobilization and in parallel loaded on to Ni(II)-bound affinity column which was equilibrated with
2
the binding buffer. Both LipT6M_CE and LipT6M_HT fractions were analyzed for protein content by
the Bradford assay (calibrated by using bovine-serum albumin) and used for soluble lipase activity
assay, i.e. the hydrolysis of pNPL. The pure lipase was eluted from the column with a linear gradient
of elution buffer (20 mM Tris–HCl buffer pH 7.5, 0.5 mM CaCl2, 0.1 mM Triton X-100, 500 mM NaCl,
and 500 mM imidazole). The eluted fractions after this step, which were defined as pure enzyme
(LipT6M fraction) were collected for immobilization and in parallel enriched with SM powder (Fluka,
Switzerland) in different ratios. The lipase concentration (%w/w) was measured with a Thermo
Scientific NanoDropTM spectrophotometer (Wilmington, DE, USA) using the lipase Mw and ε values
as 44.15 kDa and 79.3 mM-1 cm-1, respectively. These mixtures of pure lipase and SM powder were
defined as LipT6M+SM-X, where X is the weight ratio of SM powder to the lipase concentration. All
fractions from the purification process were also evaluated by SDS-PAGE analysis and pNPL
hydrolysis assay. Control experiments comprised the SM powder suspended in buffer in the
hydrolysis assay with pNPL.
1.4 Preparation of pET9a lysate
A control fraction of E. coli cells harboring pET9a vector only were prepared by culturing the cells in
0.5L TB media with 25 μg ml−1 kanamycin, harvesting the cells by centrifugation (8,000×g for 10 min
at 15°C) and resuspeding them in binding buffer. The cells were broken using a homogenizer
followed by centrifugation (16,000×g for 20 min at 15°C) for the removal of cells’ membranes. The
supernatant comprising soluble lysate proteins (termed lysate) was collected for immobilization
experiments after analysis of protein concentration and lipase activity (hydrolysis of pNPL).
2. Immobilization methods
2.1 Commercial lipases immobilization with and without pET9a crude lysate
To compare the activity of immobilized lipase T6M and the effect of the endogenous E. coli proteins
on the immobilized lipase activity, re-combined mixtures were prepared by mixing pure lipase T6M,
TLL and CaLB lipases with lysate solution. The ratio of lipase added was determined according to
the lipolytic activity (around 3 %w/w, based on the specific lipolytic activity of crude LipT6M_CE
compared to the pure enzyme). All re-combined mixtures (LipT6M+lysate, TLL+lysate, CaLB+lysate)
were immobilized in the aromatic sol gel matrix with pET9a lysate (lysate) as a bulking agent protein
mixture. Control assays comprised the pure lipases in buffer (LipT6M _3%, TLL_3%, CaLB_3%). All
the commercial solutions and the re-combined mixtures were analyzed by the Bradford assay, pNPL
hydrolysis assay and SDS-PAGE analysis. The influence of the lysate on the esterification activity,
compared to systems without lysate addition (only pure enzymes), was calculated by dividing the
activity value of the immobilized recombined mixtures of lysate-lipase, with the activity value of the
lipases immobilized with buffer only.
3
2.2 Sol-gel immobilization
Different fractions tested in this research were immobilized in a sol-gel matirx using an acidic catalyst
as published earlier (23, 24), in triplicates. In general, 27 ml glass vials were filled with 200 μL 2proapnol, 50 μL 16% PEG-1000 aqueous solution and 1 mL of the tested fraction and stirred
continuously for 10 min. The ternary precursor mixture of organosilanes (3 mmol total) were added
in the selected ratios (for aromatic matrix 2:1:1 TEOS:PTEOS:DMDEOS and for aliphatic 2:1:1
TEOS:OTEOS:DMDEOS) followed by the addition of 50 μL of 0.1 M NaF solution to initiate the solgel polymerization. After 48 h, the particles in constant stirring at 1000 rpm (Vibramax 100 orbital
shaker, Heidolph Instruments, Germany) were washed with 2-propanol, water, 2-propanol and
hexane in the exact order followed by vacuum drying at room temperature for 2 h. The dry sol-gel
particles were stored at 4ºC.
3. Activity assays
3.1 Soluble lipase activity assay
The soluble lipase hydrolytic activity was determined using a colorimetric assay as described
previously (16, 17). Briefly, the assay was based on the hydrolysis of pNPL in 96-well plates and
periodic measurement of the liberated p-nitrophenol (pNP) absorbance at 405 nm using a multi-plate
reader (Eon™ Microplate Spectrophotometer, BioTek Instruments, Inc., USA). A total of 10 μl of
lipase solution (crude or purified) was added to 180 μl reaction mixture (50mM Tris–HCl pH=8,
1.25mM CaCl2, 4% 2-propanol, and 1 % acetonitrile). The reaction was conducted at 40 °C and
started with the addition of 10 μl 20 mM pNPL dissolved in 2-propanol. Specific activity (µmol
pNPmin-1mg-1) was calculated according to the total protein concentration by Bradford assay (on
crude samples) or Nanodrop measurements (purified enzyme).
3.2 Esterification activity assay for immobilized lipase
The esterification activity of immobilized lipases, was determined by a simple esterification reaction
of n-butanol and lauric acid to obtain water and butyl-laurate, in tetraplicate. The butyl-laurate
esterification assay was based on the work of Romdhane et al. (34). The reaction started with the
addition of 50 mg of immobilized lipase to the substrate mixture (5 mmol lauric acid, 5 mmol nbutanol, 4 ml n-hexane and 50 µl water) which was equilibrated for 5 min at 45°C before the lipase
addition. The reaction was maintained at 45°C and 250 rpm in an orbital shaker for 60 min. Aliquots
(100 µl) were withdrawn periodically (every 15 min) and centrifuged (13,400 g × 2 min) to remove
the sol-gel particles. Ten µl of the upper organic layer were mixed with 490 µl of 5 mM heptadecanoic
acid methyl ester (internal standard) in ethyl-acetate for analysis in GC/MS.
4
3.2 GC/MS analysis of butyl-laurate
The butyl laurate content in the reaction mixture was analyzed and quantified using a 6890N GC
instrument (Agilent Technologies, CA, USA) equipped with a capillary HP5-MS column (60 m × 250
µm × 0.25 µm, Agilent Technologies) and 5975 Mass Selective Detector (MSD) system (Agilent
Technologies, CA, USA). One µl samples were injected in a split mode (1/20). The initial column
temperature was 150°C and kept for 3 min, raised to 240°C at 10°C min-1 and raised to 280°C at
20°C min-1 and maintained at this temperature for 1 min. The temperatures of the injector and
detector were set at 300°C. Helium was used as a carrier gas at a column flow rate of 1 ml min-1.
Under this program conditions the retention times of lauric acid, butyl laurate and methylheptadecanoate were 6.1, 8.6 and 11.2 min, respectively. Butyl laurate was quantified using a
calibration curve with butyl-laurate standard based on the ratio between its peak area and the internal
standard peak area. The activity of the dry sol-gel immobilized lipases was calculated according to
the slope comprising 5 samples throughout a 60 min reaction and expressed as µmol butyl-laurate
formed for min per gram beads (BLU/g).
4. Biodiesel production tests
4.1 Enzymatic transesterification of soybean oil and waste chicken oil by sol gel
immobilized lipase
The transesterification reactions were carried out according to the work of Dror at el. (17), in triplicate.
In general, 14 ml closed glass vials were filled with 2 g oil (soybean or waste chicken oil). Methanol
was added in 4.5:1 alcohol to oil molar ratio followed by the addition of 100 µl of lipase buffer, 5%
water content based on oil weight. Prior to immobilized lipase addition, the reaction mixture was
equilibrated at 45ºC and mixed to avoid lipase contact with high methanol concentrations. The glass
vials with the reaction mixture were maintained at 45°C in a Vibramax 100 orbital shaker (Heidolph
Instruments, Germany) at 1350 rpm. 50 µl samples were taken from the reaction mixture periodically
and centrifuged for 3 min at 20,000×g to separate the biocatalyst. 10µl aliquots of the upper organic
layer were weighted and mixed with 490 µl of ethyl acetate with 1 mg ml−1 internal standard for FAME
gas chromatographic analysis.
4.2 Gas chromatography analysis of FAME
GC analysis of FAME formation was carried out according to the work of Dror at el. (17).
4.3 Recycling experiments of sol-gel immobilized lipase (CE)
Testing the reusability potential of the sol-gel immobilized lipase was performed using the
esterification assay described above with several modifications. 200 mg of immobilized lipase were
added into the reaction mixture (2 mmol lauric acid, 4 mmol n-butanol in 4 ml n-hexane), in
tetraplicate. The reaction was maintained at 45°C and 250 rpm in an orbital shaker for 2 h. After
5
particles sedimentation, 200µl aliquots were withdrawn, centrifuged and 10 µl of the upper organic
layer were mixed with 490 µl of 5 mM internal standard solution in ethyl-acetate for analysis with
GC/MS. Conversion percentage was calculated according to the ester product and lauric acid peaks.
The reaction mixture was gently discarded (avoiding the loss of immobilized lipase) and the
remaining beads were washed 3 times with 15 ml fresh n-hexane. A fresh reaction mixture was
added to the used beads to initiate another cycle. Every two cycles the particles were dried for 1 h
at room temperature in a vacuum oven at 25C. Following the last cycle, the dried particles were
weighted and the loss of mass was calculated.
4.4 Scanning electron microscopy (SEM) analysis of sol-gel immobilized lipase
Dry sol-gel immobilized lipase particles were analyzed by scanning electron microscopy (Phenom
ProX desktop SEM). A few milligrams of dry beads were placed in the sample chamber and
visualized at room temperature.
Figure S1. Title of figure
References
6